Female NAFLD patients have significantly lower estrogen and altered gut microbiota compared to the control group
According to the lab design, females were divided into the NAFLD and control group depending on the ultrasound results for fatty liver. The results showed that there was no significant difference in age between the two groups. As shown in Figure 1A, the NAFLD group had a higher BMI and abdominal circumference, which are characteristics of obesity. However, blood glucose levels showed no statistical difference, while insulin, HOMA-IR, and triglyceride were higher in the NAFLD group, indicating the NAFLD group were insulin resistant. The average estrogen level of the NAFLD group was lower than that in the control group (74.72 ± 10.30 pg/mL vs 140.6 ± 15.76 pg/mL).
To investigate whether the gut microbiota composition differed between the two groups, we performed Illumina HiSeq 16S rRNA gene sequencing, which produced 8557224 clean reads from 45 samples. The extent of the OTUs (operational taxonomic units) shared between the two groups are summarized in the Venn diagram (Figure 1B). 617 OTUs were common, while 143 were unique to the control and 99 to the NAFLD group, revealing less OTU diversity in the NAFLD group. Rarefaction curves indicated that most gut microbial organisms in each sample were captured with the current sequencing depth (Figure 1C). As illustrated in Figure 1D, Bacteroidetes, Firmicutes, and Proteobacteria were the three dominant phyla. The abundance of Bacteroidetes and Proteobacteria exhibited significant differences between the two groups. The differences in the genera level are shown in Figure 1E. The abundance of Bacteroides, Alistipes, and Unassigned was much higher in the NAFLD group than control. LEfSe (LDA Effect Size) was used to explore significant changes and relative richness in the bacterial communities of the two groups, and five genera were identified with LDA scores (Figure 1F). Collectively,these result showed that estrogen significantly affected the composition of the gut microbiota.
Estrogen deficient mice develop severe NAFLD, and FMT attenuates NAFLD induced by estrogen deficiency.
To investigate whether estrogen deficiency is a promoter of NAFLD, mice were administrated with OVX followed by an HFD to induce NAFLD following estrogen reduction. At the same time, FMT was administered to some of the OH mice to observe whether FMT would attenuate the NAFLD (OHF group). As shown in Figure 2A, following OVX, the OH group had lower levels of estrogen than the SH group, indicating that OVX successfully reduces estrogen in animal models. Figure 2B shows the OH group weighed significantly more than the SH group, but a large reduction in body weight occurred in the OHF group. In addition, an increase in abdominal fat was found in the OH group, however, the OHF group had significantly less.
However, differences in liver mass were not found in all groups of mice. Estrogen reduction in the OH mice had clearly exacerbated fatty liver. According to the HE staining, the OH group showed more hepatic steatosis than the SH group, in that it had more lipid droplets and inflammatory cell infiltration. But the OHF group showed greater mitigation in hepatic steatosis than the OH group (Figure 2C). These results indicated that estrogen reduction would exacerbate NAFLD, but that FMT could inhibit the NAFLD in the OH mice. In addition, the NAFLD condition was assessed by the serum biochemical index. Compared with the OH group, the SH and OHF groups both had lower total cholesterol (TC) and leptin than the OH group. Serum glucose, insulin and HOMA-IR level in OH group were higher than in the SH and OHF group (Figure 2D). Although the amounts of ALT, AST and triglyceride (TG) in the OH group were slightly higher than in the SH group, no statistical differences were found. Put all together, these results indicated that estrogen reduction worsened NAFLD, while FMT could significantly relieve the condition.
The composition of the gut microbiota was changed in mice with NAFLD induced by OVX.
We then investigated whether OVX results in alterations in the composition of the gut microbiota. In total, 2,164,126 useable reads and 359 OTUs were obtained from 26 samples. The Shannon indexes were lower in the OH group compared with C and SH groups (Figure 3A). Moreover, a Venn diagram of the three groups revealed that 312 OTUs overlapped among the groups: 322 OTUs were present in both the C and SH groups; 322 in both the C and OH groups; and 323 in both the SH and OH groups (Figure 3B). PCoA (principal co-ordinates analysis) showed that gut microbiota in the C group were different from those in the SH and OH groups (Figure 3C). The system clustering tree also indicated significant differences between each group, although the distance between the SH group and the OH group was small (Figure 3D).
We further investigated the gut microbiota species and their relative abundance. As shown in Figure 3E, at the phylum level, 10 phyla could be found in all samples and the most abundant phyla in all samples were: Firmicutes, Bacteroidetes, Proteobacteria, and Epsilonbacteraeota. Conversely, Verrucomicrobia were detected in the SH and OH groups but not in the C group. Furthermore, at a genus level classification, Faecalibaculum and Helicobacter were more common in the SH and OH groups but less abundant in the C group. Interestingly, results from Figure 3F show that the presence of Muribaculaceae and Lactobacillus in the SH and OH groups were significantly decreased relative to those in the C group. Furthermore, the presence of Firmicutes was significantly lower in the OH group compared with that in the SH group; whereas the relative abundance of Epsilonbacteraeota was remarkably higher. The relative abundance of Butyricicoccus, Roseburia, and Eubacterium were diminished in the OH group compared with the SH group.
LefSe analysis was performed with the pooled data to identify specific taxa that could be used as biomarkers and dominant microbiota for each group. A cladogram for family and genus level abundance is shown in Figure 4A. In total, 41 genera were identified with LDA scores > 3.5 (Figure 4B). For correlation analysis, genera level correlations with SCFA and serological indicators such as Estrogen, TC, and leptin are shown in Figures 4C and 4D. Peptococcus and Romboutsia were positively, while Ruminiclostridiun-6 and Muribaculum were negatively correlated with SCFA. The occurrence of the genera exhibited a positive correlation with TC, leptin and body weight. Surprisingly, eetrogen showed negative correlations with most of the genera detected. Put all together, our results showed that estrogen could modulate gut microbiota composition in NAFLD model mice.
Butyrate is decreased in NAFLD patients and in the OH mice, andsupplementation with butyrate inhibits NAFLD caused by estrogen reduction.
To determine whether SCFA was changed in NAFLD patients with decreased estrogen, the concentration of their SCFA was examined. The butyrate content was much lower in the NAFLD patients than that in the controls (Figure 5A). However, the concentration of other SCFAs showed no difference between the two groups. Butyrate was also significantly decreased in the OH mice compared with the SH group (Figure 5B). Consistently, the other SCFAs showed no change. The content profile of SCFAs in OH mice was similar to that found in the NAFLD patients with decreased estrogen. These results indicate that the butyrate content is affected by estrogen in the NAFLD model.
To determine whether decreased butyrate causes the NAFLD according to estrogen reduction, the OH group was given butyrate(200mg/kg)for 4 weeks (named the OHB group). Results showed that the OHB group had no statistical difference in body and abdominal fat mass compared with the OH group, but after receiving the butyrate complement, their liver weight was largely decreased (Figure 5C). HE staining also demonstrated that hepatocyte steatosis was reduced in the OHB group (Figure 5D). Furthermore, serum biochemical indicators also showed lower serum TC, leptin, blood glucose, and insulin in OHB mice, but no statistical differences were shown between the groups for ALT and AST (Figure 5E).
Estrogen reduced mice have lower production of antimicrobial peptide (AMP) in IEC.
We then investigated whether the altered microbiota and butyrate levels in estrogen reduced mice affect IEC function. AMP produced by IEC of Paneth cells play a critical role in regulation of host and microbiota interaction. In order to study AMP production, IEC were collected to determine their AMP expression. Based on the results from qRT-PCR, the OH group had decreased expression of Reg3γ, β-defensins 1 and 3 than the SH group at mRNA levels (Figure 6A), and protein level of Reg3γ and β-defensins 1 were also decreased (Figure 6B and C). However, no difference was detected between the two groups for β-defensins 4. These results confirmed that AMP production was decreased in mice with NAFLD caused by estrogen reduction.
Expression of intestinal epithelial tight junction related genes and proteins are changed in estrogen reduced mice.
The mechanism by which hepatic steatosis is induced by estrogen reduction was studied. Because the integrity of the intestinal epithelium plays a crucial role in maintaining a balance in fatty acid intake, damage to the intestinal epithelial barrier leads to an increase in fatty acid intake and potentially to fatty liver disease. The barrier function of the intestinal epithelium were investigated. The expression of the ZO-1 and Occludin5 genes were lower in the OH group than in the SH group (Figure 7A), and expression of the protein Occludin5 had also significantly declined in the OH group (Figure 7B). In addition, HE staining showed that the OH group had reduced number of epithelium cells nucleus,with thickening of the intestinal wall (Figure 7C). The results confirmed that the intestinal epithelial barrier function was damaged in estrogen reduced mice.
Fatty acid (FA) synthesis, intake, and oxidation related gene and protein expression are changed in estrogen reduced mice.
As the central organ in the metabolic reactions, the liver plays an important role in maintaining metabolic balance. NAFLD is a disease expressed through multiple metabolic disorders and especially in FA metabolism. We hypothesize that mice with an estrogen reduction have a disorder of FA metabolism pathway. Therefore, genes and protein related to FA metabolism were tested in these mice. The expression of the lipid synthesis-related genes, SREBP1, PPAR-ɣ, FAS and CHREB were found to be significantly increased with estrogen reduction (Figure 8A). Moreover, in comparison with the SH group mice, the OH group had a significantly greater expression of the lipid intake related gene VLDLR (Figure 8B), while the lipid oxidation related genes, PPAR-ɑ and ACAA, had lower expression in the OH group (Figure 8C). The Western blot shown that the OH group mice had increases in PPAR-ɣ and VLDLRB proteins but PPAR-ɑ expression had decreased (Figure 8D). In summary, estrogen was shown to be a crucial factor in FA metabolism regulation in the liver.